Abstract
Objective
Rare variants in the TREM2 gene have been reported to significantly increase the risk of Alzheimer's disease in Caucasian populations. Hitherto, this association was not studied in North African populations. In this work, we aimed to study the association between TREM2 exon 2 variants and the risk of late-onset Alzheimer's disease (LOAD) in a Tunisian population.
Subjects and Methods
We sequenced exon 2 of TREM2 in a Tunisian cohort of 172 LOAD patients and 158 control subjects. We used the Fisher exact test to compare the distribution of allelic frequencies between the two groups.
Results
We identified 4 previously reported nonsynonymous variants (p.Asp39Glu, p.Arg62His, p.Thr96Lys, and p.Val126Gly) and 1 novel synonymous variant (p.Gln109Gln), none of which was significantly associated with the risk of Alzheimer's disease. Moreover, the rare TREM2 variant (p.Arg47His), which was considered to be a risk factor for Alzheimer's disease in European descent populations, was not detected in our cohort.
Conclusion
These findings do not support a major role for TREM2 in the pathogenesis of LOAD in the Tunisian population.
Keywords: TREM2 gene, Variants, Alzheimer's disease, North-African population, Case-control study
Significance of the Study
Rare variants within exon 2 of the TREM2 gene increase the risk of Alzheimer's disease in Caucasian populations. This is the first case-control study to assess the association between these variants and the risk of Alzheimer's disease in a North African population. We sequenced exon 2 of the TREM2 gene in a cohort of Tunisian patients with late-onset Alzheimer's disease and healthy individuals and identified 5 variants, none of which was associated with the risk of Alzheimer's disease. Our study does not support a major role for TREM2 in the pathogenesis of late-onset Alzheimer's disease in the Tunisian population.
Introduction
TREM2 gene encodes the triggering receptor expressed on myeloid cells 2, which is highly expressed in microglia of the central nervous system [1] and is involved in regulating the immune system by promoting phagocytosis and regulating the inflammatory response. The rare missense mutation p.Arg47His (rs75932628) within TREM2 increases the risk of neurodegenerative disorders such as Parkinson's disease [2], essential tremor [3], and late-onset Alzheimer's disease (LOAD). This mutation was identified as a rare risk factor for LOAD in several European descent cohorts with an odds ratio similar to that for apolipoprotein E epsilon 4 (ApoE ε4) [4, 5].
TREM2 variants associated with Alzheimer's disease (AD) have been screened in various populations worldwide; however, no studies have been carried-out in North African populations. Most of the aforementioned variants were observed in exon 2 of the TREM2 gene. Therefore, the aim of this work was to evaluate the association of TREM2 exon 2 variants with risk of AD in a sample of the Tunisian population.
Subjects and Methods
Subjects
One hundred seventy-two Tunisian patients with LOAD were recruited from the Neurology Department of Razi Hospital, Manouba, Tunisia. Clinical diagnosis of LOAD was done according to the criteria of the National Institute of Neurological and Communication Disorders and Stroke-Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) [6]. In addition, 158 unrelated control subjects were recruited from different primary care clinics. None of the control group subjects had cognitive impairment or personal or familial history of neurological and psychiatric disorders. Written informed consent was obtained from each individual prior to enrollment in the genetic study. Research protocols were approved by the Medical Ethics Committee of Razi Hospital and conformed to the guidelines of the Declaration of Helsinki.
ApoE Genotyping and TREM2 Sequencing
Blood samples were collected from LOAD patients and controls. Genomic DNA was extracted from blood by the salting-out method [7]. ApoE genotyping was performed as previously described [8]. Exon 2 of TREM2 gene was amplified by polymerase chain reaction (PCR) from genomic DNA using two primers: 5′-TGAATGAATGTCTCCTCCCCAG-3′ and 5′-CAGCCACTGCCCACTCA-3′, under the following reaction conditions: denaturation at 95°C for 5 min followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and a final cycle of 7-min extension at 72°C. PCR products were purified and sequenced using the BigDye Terminator Cycle Sequencing Kit v1.1 on a 3,500xl Genetic Analyzer DNA sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed using the SeqScape software (Applied Biosystems) and compared to the TREM2 GenBank reference sequence (NM_018965).
In silico Tools
The impact of TREM2 variations was predicted using SIFT (Sorting Intolerant From Tolerant; http://sift.jcvi.org/www/SIFT_chr_coords_submit.html), PolyPhen-2 (Polymorphism Phenotyping v2; http://genetics.bwh.harvard.edu/pph2/) and PredictSNP2 (http://loschmidt.chemi.muni.cz/predictsnp2/) [9]. Frequencies of variants were reported from exome database (1,000G; http://browser.1000genomes.org/index.html), ExAC (http://exac.broadinstitute.org/), EVS (http://evs.gs.washington.edu/EVS/), and Great Middle East (GME) Variome Project database (http://igm.ucsd.edu/gme/data-browser.php) including 508 samples from North Africa.
Statistical Analysis
We used Fisher's exact test to compare the distribution of allelic frequencies between LOAD patients and control groups. ApoE genotype and sex distributions were compared using the χ2 test and mean age was compared using the t test. All analyses were two-tailed and a p value of 0.05 or less was considered statistically significant. Odds ratios and 95% confidence intervals were calculated using SPSS.
Results
Demographic characteristics and ApoE genotypes of Tunisian LOAD patients (n = 172) and controls (n = 158) are summarized in Table 1. Both groups had similar sex ratios and similar mean ages. ApoE genotyping showed a significant difference in genotype distributions between the two groups; as expected, the ApoE ε4 allele was overrepresented in LOAD patients compared to the control group (36.9 vs. 16.1%; p < 0.05). The ApoE ε2 allele was not detected in patients and controls.
Table 1.
Demographic characteristics of subjects
| LOAD patients | Controls | p value | |
|---|---|---|---|
| Subjects, n | 172 | 158 | |
| Sex ratio (male:female) | 0.5 (58:114) | 0.64 (62:96) | 0.297 |
| Mean age at examination ± SD, years | 75.84±9.64 | 74.27±4.17 | 0.06 |
| Mean age at onset ± SD, years | 68.69±10.05 | ||
| ApoE ε4/ApoE ε4, n (%) | 27 (15.7) | 3 (1.9) | 1.3 10e-5 |
| ApoE ε3/ApoE ε4, n (%) | 73 (42.4) | 45 (28.5) | 0.008 |
| ApoE ε3/ApoE ε3, n (%) | 72 (41.9) | 110 (69.6) | 0.03 |
LOAD, late-onset Alzheimer's disease; SD, standard deviation.
Sequencing of exon 2 of the TREM2 gene revealed 5 variants in 14 LOAD patients and 1 variant in 5 controls. As shown in Table 2, 4 previously reported nonsynonymous variants (p.Asp39Glu, p.Arg62His, p.Thr96Lys, and p.Val126Gly) and 1 novel synonymous variant (p.Gln109Gln) were observed in patients. The variant p.Thr96Lys was also identified in controls. All variants were present in the heterozygous state and were detected in separate individuals (no individuals carried 2 or more variants). Among the 14 patients carrying the TREM2 variants, 11 had a positive family history of AD (Table 3).
Table 2.
Variants of TREM2 after sequencing of exon 2 in LOAD patients and controls
| Variant | db SNP ID | Position (GRCh37) | Exonic function | LOAD patients (carriers, MAF) | Controls (carriers, MAF) | p value | OR (95% CI) | Population frequency data |
|||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1,000G | gnomAD | EVS | GME | ||||||||
| p.Asp39Glu | rs200392967 | g.41129275G>C | nonsyn | 1 (0.005) | 0 | 1 | NA | 0 | 6.5 e-5 | 7.6 e-5 | 0 |
| p.Arg62His | rs143332484 | g.41129207C>T | nonsyn | 2 (0.011) | 0 | 0.49 | NA | 0.005 | 0.008 | 0.007 | 0 |
| p.Thr96Lys | rs2234253 | g.41129105G>T | nonsyn | 5 (0.029) | 6 (0.037) | 0.76 | 1.3 (0.39–4.36) |
0.041 | 0.012 | 0.039 | 0 |
| p.Gln109Gln | NA | g.41129065C>T | syn | 5 (0.029) | 0 | 0.06 | NA | ||||
| p.Val126Gly | rs121908402 | g.41129015A>C | nonsyn | 1 (0.005) | 0 | 1 | NA | 0 | 8.2 e-6 | 0 | 0 |
LOAD, late-onset Alzheimer's disease; db SNP, single nucleotide polymorphism database; MAF, minor allele frequency; syn, synonymous; nonsym, nonsynonymous.
Table 3.
Characteristics of individuals carrying the TREM2 variants
| Variant | Carrier | Gender | Age at examination/Age at onset, years | ApoE genotype | Family history of neurodegenerative disease |
|---|---|---|---|---|---|
| p.Asp39Glu | patient 1 | F | 86/82 | ε4/ε4 | yes |
| p.Arg62His | patient 2 | M | 79/71 | ε3/ε3 | yes |
| patient 3 | F | 78/71 | ε3/ε3 | yes | |
| p.Thr96Lys | patient 4 | M | 79/66 | ε4/ε4 | yes |
| patient 5 | M | 69/67 | ε3/ε3 | yes | |
| patient 6 | F | 72/65 | ε3/ε4 | yes | |
| patient 7 | F | 80/75 | ε3/ε4 | yes | |
| patient 8 | F | 89/79 | ε3/ε3 | yes | |
| control 1 | M | 71/– | ε3/ε3 | no | |
| control 2 | M | 68/– | ε3/ε4 | no | |
| control 3 | F | 70/– | ε3/ε3 | no | |
| control 4 | M | 68/– | ε3/ε3 | no | |
| control 5 | F | 70/– | ε3/ε4 | no | |
| p.Gln109Gln | patient 9 | M | 85/70 | ε3/ε4 | yes |
| patient 10 | F | 75/65 | ε4/ε4 | yes | |
| patient 11 | F | 72/67 | ε3/ε3 | no | |
| patient 12 | F | 85/68 | ε3/ε4 | no | |
| patient 13 | F | 77/72 | ε3/ε3 | no | |
| p.Val126Gly | patient 14 | M | 82/72 | ε3/ε4 | yes |
The frequencies of each variation in the population's exome sequencing database are detailed in Table 2. No common variants having minor allele frequency (MAF) > 5% were found, and 2 variants (p.Val126Gly and p.Asp39Glu) were rare, with MAF < 1%. Three variants were predicted to have a probable damaging effect (p.Asp39Glu, p.Thr96Lys, and p.Val126Gly; Table 4). The association analysis of all the identified TREM2 variants and AD was not statistically significant (Table 2). Even the rare variants p.Val126Gly and p.Asp39Glu were not significantly associated with AD (p > 0.05).
Table 4.
In silico prediction of TREM2 missense variants
| Variant | db SNP ID | Position (GRCh37) | SIFT | Polyphen2 | PredictSNP2 |
|---|---|---|---|---|---|
| p.Asp39Glu | rs200392967 | g.41129275G>C | tolerated | possibly damaging | deleterious |
| p.Arg62His | rs143332484 | g.41129207C>T | tolerated | benign | neutral |
| p.Thr96Lys | rs2234253 | g.41129105G>T | damaging | probably damaging | neutral |
| p.Val126Gly | rs121908402 | g.41129015A>C | damaging | Probably damaging | deleterious |
Discussion
This is the first investigation into the possible association between TREM2 variants and risk of AD in a North African population. In this study, we sequenced exon 2 of the TREM2 gene in a cohort of Tunisian patients with LOAD and healthy individuals, and identified 5 variants (p.Asp39Glu, p.Arg62His, p.Thr96Lys, p.Gln109Gln, and p.Val126Gly). The p.Arg47His mutation was not found in our cohort. Association analysis revealed that none of the identified variants were associated with AD risk. Therefore, we could not test the interaction of the ApoE ε4 allele with these variants.
A summary of the reported screening studies for TREM2 mutations to date, including AD patients, is presented in Table 5. TREM2 p.Arg62His was previously observed in AD patients and controls in Spanish, Belgian, and French populations (Table 5), and did not show a nominally significant association with AD. However, a previous case-control study of large European American descent cohorts showed that in addition to p.Arg47His [10], p.Arg62His is a risk factor for AD. The variant rs2234253 (p.Thr96Lys), detected in Belgian, French, and Japanese populations (Table 5), was not shown to enhance AD risk. In our study, p.Thr96Lys was the unique variant detected in controls and LOAD patients with equivalent MAF. According to public exome databases, this variant was more common in African populations (MAF = 14.5%, 1,000G) than other populations, but was not present in North African populations of the GME (Table 2).
Table 5.
Summary of all variant screening studies of TREM2 in patients with Alzheimer's disease (AD)
| Ethnicity | AD patients |
TREM2 variants in AD patients |
||
|---|---|---|---|---|
| exon 2 | exon 3 | exon 4 | ||
| Caucasian | 256 European + 836 North American [5] |
Q33X, Y38C, R47H, R62H, T66M, D87N, T96K, R98W |
R136Q, H157Y | - |
| 117 Norwegian + 944 Dutch + | R47H | - | - | |
| 517 German + 3,759 Icelandic + | ||||
| 399 US [4] | ||||
| 726 French [13] | Q33X, R47H, R62H, R62C, D87N, T96K | - | - | |
| 427 American [14] | R47H | - | - | |
| 504 Spanish [15] | R47H, R62H | - | - | |
| 3,172 Spanish [16] | R47H | - | - | |
| 1,216 Belgian [11] | D39G, D39E, R47H, G58A, R62H, D87N, T96K |
L133L, H157Y | S162R, L211P, T223I |
|
| 2,082 US [10] | Q33X, R47H, R52H, R62H, T66M, D87N, T96K |
R136W, R136Q, H157Y |
W191X, E202D, L211P, H215Q, T223I |
|
| 210 North American [17] | R47H, D87N, | H157Y | L205P, G219C | |
| East Asian | 1,133 Chinese [18] | V34V, C110C, H114H, G115S | - | - |
| 360 Chinese [19] | A130V | - | - | |
| 988 Chinese [20] | - | H157Y | S183C, A192T | |
| 400 Korean [21] | - | H157Y | A192T | |
| 2,190 Japanese [22] | R47H, | H157Y | L211P | |
| African American | 899 African American [23] | R47H, R62H, D87N | E151K, | W191X, L211P |
| Others | 131 Iranian [24] | R47H, G55R, R62H, R62C | - | - |
Variations significantly associated with AD are underlined.
Two rare variants predicted to be deleterious, p.Asp39Glu and p.Val126Gly, were identified in our study. The p.Asp39Glu was previously reported in 2 AD patients at the heterozygous state [11], while homozygous p.Val126Gly was reported in 2 patients with Nasu-Hakola disease [12]. Among the 5 variants, variant Gln109Gln was not found in any of the exome databases. However, this variant is synonymous and does not change the amino acid sequence of TREM2 protein.
The previously reported rare variant rs75932628 (p.Arg47His), identified to be a risk factor for AD in Caucasian populations (Table 5), was not found in our cohort. Our findings are in agreement with published reports of African American, Iranian, and East Asian populations (Table 5) which showed that the p.Arg47His mutation could not be linked to an increased risk of AD. In our study, the lack of association of the p.Arg47His variant may be due to its very low MAF in the Tunisian population. In the GME database, this genetic polymorphism had a global MAF of 0.15 and 0.1% in North African populations. Moreover, this variant is considered to be a risk factor according to the ethnicity of the population. AD risk in the Tunisian population may be influenced by genetic and/or environmental factors which might reduce the effect of this variant. However, the limitation of our work is the small cohort size, which could explain the lack of statistical significance.
Conclusion
This study is the first to explore the possibility of an association of TREM2 with the risk of AD in North Africa, particularly in Tunisia. This population seems to be closer to the African American or East Asian populations than to the European ones regarding the AD risk factor p.Arg47His. We hypothesize that variations in exon 2 of TREM2 may not play a major role in the pathogenesis of LOAD in the Tunisian population. However, further studies on larger cohorts of North African populations are needed to confirm this hypothesis.
Disclosure Statement
The authors declare that they have no conflicts of interest.
Acknowledgements
We are grateful to all the patients and their family members for participating in this study. This research was supported by grants from the Tunisian Ministry of Higher Education and Scientific Research and the Tunisian Ministry of Health.
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